Upregulation of LHPP by saRNA inhibited hepatocellular cancer cell proliferation and xenograft tumor growth.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer worldwide and no pharmacological treatment is available that can achieve complete remission of HCC. Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) is a recently identified HCC tumor suppressor gene which plays an important role in the development of HCC and its inactivation and reactivation has been shown to result in respectively HCC tumorigenesis and suppression. Small activating RNAs (saRNAs) have been used to achieve targeted activation of therapeutic genes for the restoration of their encoded protein through the RNAa mechanism. Here we designed and validated saRNAs that could activate LHPP expression at both the mRNA and protein levels in HCC cells. Activation of LHPP by its saRNAs led to the suppression of HCC proliferation, migration and the inhibition of Akt phosphorylation. When combined with targeted anticancer drugs (e.g., regorafenib), LHPP saRNA exhibited synergistic effect in inhibiting in vitro HCC proliferation and in vivo antitumor growth in a xenograft HCC model. Findings from this study provides further evidence for a tumor suppressor role of LHPP and potential therapeutic value of restoring the expression of LHPP by saRNA for the treatment of HCC.

Puerarin improves busulfan-induced disruption of spermatogenesis by inhibiting MAPK pathways.

Male infertility is a global concern, with a noticeable increase in the decline of spermatogenesis and sperm quality. However, there are limited clinically effective treatments available. This study aimed to investigate the potential effectiveness of puerarin in treating male infertility, which leads to gonadal changes. The results obtained from various analyses such as CASA, immunofluorescence, DIFF-Quick, hematoxylin and eosin (H&E), and periodic acid-Schiff (PAS) staining demonstrated that puerarin supplementation significantly alleviated the busulfan-induced reduction in spermatogenesis and sperm quality in both young and adult mice. Furthermore, puerarin exhibited a marked improvement in the damage caused by busulfan to the architecture of seminiferous tubules, causal epididymis, blood-testicular barrier (BTB), as well as spermatogonia and Sertoli cells. Similarly, puerarin significantly reduced the levels of total antioxidant capacity (T-AOC), malondialdehyde (MDA), and caspase-3 in the testes of busulfan-induced mice, as determined by microplate reader analysis. Additionally, RNA-seq data, RT-qPCR, and western blotting revealed that puerarin restored the abnormal gene expressions induced by busulfan to nearly healthy levels. Notably, puerarin significantly reversed the impact of busulfan on the expression of marker genes involved in spermatogenesis and oxidative stress. Moreover, puerarin suppressed the phosphorylation of p38, ERK1/2, and JNK in the testes, as observed through testicular analysis. Consequently, this study concludes that puerarin may serve as a potential alternative for treating busulfan-induced damage to male fertility by inactivating the testicular MAPK pathways. These findings may pave the way for the use of puerarin in addressing chemotherapy- or other factors-induced male infertility in humans.

Hydrogen sulfide protects Sertoli cells against toxicant Acrolein-induced cell injury.

Acrolein (ACR), a highly toxic α,ß-unsaturated aldehyde, is considered to be a common mediator behind the reproductive injury induced by various factors. However, the understanding of its reproductive toxicity and prevention in reproductive system is limited. Given that Sertoli cells provide the first-line defense against various toxicants and that dysfunction of Sertoli cell causes impaired spermatogenesis, we, therefore, examined ACR cytotoxicity in Sertoli cells and tested whether hydrogen sulfide (H2S), a gaseous mediator with potent antioxidative actions, could have a protective effect. Exposure of Sertoli cells to ACR led to cell injury, as indicated by reactive oxygen species (ROS) generation, protein oxidation, P38 activation and ultimately cell death that was prevented by antioxidant N-acetylcysteine (NAC). Further studies revealed that ACR cytotoxicity on Sertoli cells was significantly exacerbated by the inhibition of H2S-synthesizing enzyme cystathionine γ-lyase (CSE), while significantly suppressed by H2S donor Sodium hydrosulfide (NaHS). It was also attenuated by Tanshinone IIA (Tan IIA), an active ingredient of Danshen that stimulated H2S production in Sertoli cells. Apart from Sertoli cells, H2S also protected the cultured germ cells from ACR-initiated cell death. Collectively, our study characterized H2S as endogenous defensive mechanism against ACR in Sertoli cells and germ cells. This property of H2S could be used to prevent and treat ACR-related reproductive injury.

Puerarin extends the lifespan of Drosophila melanogaster by activating autophagy.

Lifespan longevity has attracted increasing attention with societal development. To counter the effects of aging on longevity, we focused on the natural chemicals of plants. In this study, we investigated the effects of puerarin supplementation on the lifespan of Drosophila melanogaster. Puerarin supplementation significantly extended the lifespan of D. melanogaster at 60 µM and 120 µM by upregulating proteasome subunit beta 5 (prosbeta5) and sirtuin-1 (Sirt1). However, puerarin-induced longevity of male flies (F0 generation) may not be passed on to descendants. Additionally, a puerarin diet for 10 and 25 days did not influence the body weight and food intake of male Canton-S flies. Puerarin significantly improved the climbing ability, starvation resistance, and oxidation resistance of male flies by upregulating the expression of Shaker, catalase (CAT), superoxide dismutase 1 (SOD1), and Methuselah, and downregulating poly [ADP-ribose] polymerase (PARP-1) and major heat shock 70 kDa protein Aa (HSP70). Moreover, 120 µM puerarin supplementation for 25 days significantly increased adenosine 5' triphosphate (ATP) content by increasing adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) levels. Additionally, the puerarin diet for 25 days suppressed male fecundity in male flies by decreasing the levels of Bam and Punt. Mechanistically, puerarin enhanced lysosome-involved autophagy by promoting the expression of lysosome markers [ß-galactosidase and lysosomal associated membrane protein 1 (LAMP1)], and elevating the levels of autophagy-related genes, including autophagy-associated gene 1 (ATG1), ATG5, and ATG8b. However, puerarin decreased the phosphorylation of the target of rapamycin (TOR) protein. In conclusion, puerarin is a promising compound for improving the longevity of D. melanogaster by activating autophagy.

Single-Cell Transcriptomics Reveals Longevity Immune Remodeling Features Shared by Centenarians and Their Offspring.

Centenarians, who show mild infections and low incidence of tumors, are the optimal model to investigate healthy aging. However, longevity related immune characteristics has not been fully revealed largely due to lack of appropriate controls. In this study, single-cell transcriptomic analysis of peripheral blood mononuclear cells (PBMCs) derived from seven centenarians (CEN), six centenarians' offspring (CO), and nine offspring spouses or neighbors (Control, age-matched to CO) are performed to investigate the shared immune features between CEN and CO. The results indicate that among all 12 T cell clusters, the cytotoxic-phenotype-clusters (CPC) and the naïve-phenotype-clusters (NPC) significantly change between CEN and ontrol. Compared to Control, both CEN and CO are characterized by depleted NPC and increased CPC, which is dominated by CD8+ T cells. Furthermore, CPC from CEN and CO share enhanced signaling pathways and transcriptional factors associated with immune response, and possesse similar T-cell-receptor features, such as high clonal expansion. Interestingly, rather than a significant increase in GZMK+ CD8 cells during aging, centenarians show accumulation of GZMB+ and CMC1+ CD8 T cells. Collectively, this study unveils an immune remodeling pattern reflected by both quantitative increase and functional reinforcement of cytotoxic T cells which are essential for healthy aging.

Structures of the ADGRG2-Gs complex in apo and ligand-bound forms.

Adhesion G protein-coupled receptors are elusive in terms of their structural information and ligands. Here, we solved the cryogenic-electron microscopy (cryo-EM) structure of apo-ADGRG2, an essential membrane receptor for maintaining male fertility, in complex with a Gs trimer. Whereas the formations of two kinks were determinants of the active state, identification of a potential ligand-binding pocket in ADGRG2 facilitated the screening and identification of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate and deoxycorticosterone as potential ligands of ADGRG2. The cryo-EM structures of DHEA-ADGRG2-Gs provided interaction details for DHEA within the seven transmembrane domains of ADGRG2. Collectively, our data provide a structural basis for the activation and signaling of ADGRG2, as well as characterization of steroid hormones as ADGRG2 ligands, which might be used as useful tools for further functional studies of the orphan ADGRG2.

Physiologically detectable bisphenol A impairs human sperm functions by reducing protein-tyrosine phosphorylation.

BACKGROUND: Bisphenol A (BPA), a widely used plastic monomer and plasticizer, is detectable in blood, urine and semen of a healthy people, with concentrations ranging from 0.1 nM to 10 nM. It has been shown that in vitro exposure of BPA as low as 0.001 nM could significantly inhibited mouse sperm motility and acrosome reaction. However, it is still unclear whether BPA at those physiologically detectable concentration affects human sperm. METHODS: The effects of different concentrations of BPA (0, 10-3, 10-2, 10-1, 10, 103 nM) on sperm functions were examined, including human sperm viability, kinematic parameters, hyperactivation and capacitation. RESULTS: BPA caused a remarkable decline in human sperm viability, motility and progressive motility, hyperactivation, capacitation and progesterone-induced acrosome reaction. Mechanism studies showed that BPA could suppress the protein tyrosine phosphorylation level of human sperm, but had no effect on sperm calcium signaling. CONCLUSIONS: Physiologically detectable concentrations of BPA may impair human sperm functions via suppressing protein tyrosine phosphorylation of human sperm, implying that environmental pollution of BPA might be a factor contributing to male infertility.

Na+/H+ Exchangers Involve in Regulating the pH-Sensitive Ion Channels in Mouse Sperm.

Sperm-specific K+ ion channel (KSper) and Ca2+ ion channel (CatSper), whose elimination causes male infertility in mice, determine the membrane potential and Ca2+ influx, respectively. KSper and CatSper can be activated by cytosolic alkalization, which occurs during sperm going through the alkaline environment of the female reproductive tract. However, which intracellular pH (pHi) regulator functionally couples to the activation of KSper/CatSper remains obscure. Although Na+/H+ exchangers (NHEs) have been implicated to mediate pHi in sperm, there is a lack of direct evidence confirming the functional coupling between NHEs and KSper/CatSper. Here, 5-(N, N-dimethyl)-amiloride (DMA), an NHEs inhibitor that firstly proved not to affect KSper/CatSper directly, was chosen to examine NHEs function on KSper/CatSper in mouse sperm. The results of patch clamping recordings showed that, when extracellular pH was at the physiological level of 7.4, DMA application caused KSper inhibition and the depolarization of membrane potential when pipette solutions were not pH-buffered. In contrast, these effects were minimized when pipette solutions were pH-buffered, indicating that they solely resulted from pHi acidification caused by NHEs inhibition. Similarly, DMA treatment reduced CatSper current and intracellular Ca2+, effects also dependent on the buffer capacity of pH in pipette solutions. The impairment of sperm motility was also observed after DMA incubation. These results manifested that NHEs activity is coupled to the activation of KSper/CatSper under physiological conditions.

The Ca2+ channel CatSper is not activated by cAMP/PKA signaling but directly affected by chemicals used to probe the action of cAMP and PKA.

The sperm-specific Ca2+ channel CatSper (cation channel of sperm) controls the influx of Ca2+ into the flagellum and, thereby, the swimming behavior of sperm. A hallmark of human CatSper is its polymodal activation by membrane voltage, intracellular pH, and oviductal hormones. Whether CatSper is also activated by signaling pathways involving an increase of cAMP and ensuing activation of PKA is, however, a matter of controversy. To shed light on this question, we used kinetic ion-sensitive fluorometry, patch-clamp recordings, and optochemistry to study transmembrane Ca2+ flux and membrane currents in human sperm from healthy donors and from patients that lack functional CatSper channels. We found that human CatSper is neither activated by intracellular cAMP directly nor indirectly by the cAMP/PKA-signaling pathway. Instead, we show that nonphysiological concentrations of cAMP and membrane-permeable cAMP analogs used to mimic the action of intracellular cAMP activate human CatSper from the outside via a hitherto-unknown extracellular binding site. Finally, we demonstrate that the effects of common PKA inhibitors on human CatSper rest predominantly, if not exclusively, on off-target drug actions on CatSper itself rather than on inhibition of PKA. We conclude that the concept of an intracellular cAMP/PKA-activation of CatSper is primarily based on unspecific effects of chemical probes used to interfere with cAMP signaling. Altogether, our findings solve several controversial issues and reveal a novel ligand-binding site controlling the activity of CatSper, which has important bearings on future studies of cAMP and Ca2+ signaling in sperm.

Lysine glutarylation in human sperm is associated with progressive motility.

STUDY QUESTION: Is there a role for lysine glutarylation (Kglu), a newly identified protein post-translational modification (PTM), in human sperm? SUMMARY ANSWER: Kglu occurs in several proteins located in the tail of human sperm, and it was reduced in asthenozoospermic (A) men and positively correlated with progressive motility of human sperm, indicating its important role in maintaining sperm motility. WHAT IS KNOWN ALREADY: Since mature sperm are almost transcriptionally silent, PTM is regarded as an important pathway in regulating sperm function. However, only phosphorylation has been extensively studied in mature sperm to date. Protein lysine modification (PLM), a hot spot of PTMs, was rarely studied except for a few reports on lysine methylation and acetylation. As a newly identified PLM, Kglu has not been well characterized, especially in mature sperm. STUDY DESIGN, SIZE, DURATION: Sperm samples were obtained from normozoospermic (N) men and A men who visited the reproductive medical center between February 2016 and January 2018. In total, 61 N men and 59 A men were recruited to participate in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Kglu was examined by immunoblotting and immunofluorescence assays using a previously qualified pan-anti-glutaryllysine antibody that recognizes glutaryllysine in a wide range of sequence contexts (both in histones and non-histone substrates) but not the structurally similar malonyllysine and succinyllysine. The immunofluorescence assay was imaged using laser scanning confocal microscopy and super-resolution structured illumination microscopy. Sperm motility parameters were examined by computer-assisted sperm analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Kglu occurs in several proteins (20-150 kDa) located in the tail of human sperm, especially in the middle piece and the latter part of the principal piece. Sperm Kglu was modulated by regulatory systems (enzymes and glutaryl-CoA) similar to those in HeLa cells. The mean level of sperm Kglu was significantly reduced in A men compared with N men (P < 0.001) and was positively correlated with progressive motility (P < 0.001). The sodium glutarate-induced elevation of Kglu levels in A men with lower Kglu levels in sperm significantly improved the progressive motility (P < 0.001). Furthermore, the reduced sperm Kglu levels in A men was accompanied by an increase in sperm glutaryl-CoA dehydrogenase (a regulatory enzyme of Kglu). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Although the present study indicated the involvement of sperm Kglu in maintaining progressive motility of human sperm, the underlying mechanism needs to be investigated further. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this study provide an insight into the novel role of Kglu in human sperm and suggest that abnormality of sperm PLMs may be one of the causes of asthenozoospermia. STUDY FUNDING/COMPETING INTEREST(S): National Natural Science Foundation of China (81 771 644 to T.L.; 31 671 204 to X.Z. and 81 871 207 to H.C.); National Basic Research Program of China (973 Program, 2015CB943003 to X.Z.); Natural Science Foundation of Jiangxi, China (20171ACB21006 and 20161BAB204167 to T.L.; 20165BCB18001 to X.Z.). The authors have no conflicts of interest to declare.

A novel copy number variation in CATSPER2 causes idiopathic male infertility with normal semen parameters.

STUDY QUESTION: Are genetic abnormalities in CATSPER (cation channel of sperm) genes associated with idiopathic male infertility with normal semen parameters and, if so, how do they affect male fertility? SUMMARY ANSWER: A novel copy number variation (CNV) in CATSPER2 causes idiopathic male infertility with normal semen parameters by disrupting the ability of sperm to penetrate viscous media, undergo hyperactivation and respond to progesterone. WHAT IS KNOWN ALREADY: CATSPER is the principle Ca2+ channel mediating extracellular Ca2+ influx into spermatozoa. Although several case reports have suggested a causal relationship between CATSPER disruption and human male infertility, whether genetic abnormalities in CATSPER genes are associated with idiopathic male infertility with normal semen parameters remains unclear. STUDY DESIGN, SIZE, DURATION: Spermatozoa were obtained from men attending the reproductive medical center at Jiangxi Provincial Maternal and Child Health Hospital, Nanchang, Jiangxi, China between January 2014 and June 2016. In total, 120 men from infertile couples and 20 healthy male donors were selected to take part in the study, based on their normal semen parameters. PARTICIPANTS/MATERIALS, SETTING, METHODS: CATSPER and KSPER currents were assessed using the whole-cell patch-clamp technique. Whole-genome sequencing and TaqMan® CNV assays were performed to identify genetic variations. The expression levels of genes encoding the CATSPER complex were measured by quantitative real-time PCR and Western blot. Sperm motion characteristics and hyperactivation were examined with a computer-aided sperm analysis (CASA) system. Sperm responses to progesterone, assessed as increases in CATSPER current and intercellular Ca2+ concentrations ([Ca2+]i), as well as inducement of penetration ability and acrosome reaction, were examined by means of whole-cell patch-clamp technique, single-sperm [Ca2+]i imaging, penetration into methylcellulose assay and chlortetracycline staining, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: An infertile man with complete disruption of CATSPER current was identified. This individual has a novel CNV which disrupts one gene copy in the region 43894500-43950000 in chromosome 15 (GRCh37.p13 Primary Assembly, nsv3067119), containing the whole DNA sequence of CATSPER2. This CNV affected the expression of CATSPER2, resulting in dramatically reduced levels of CATSPER2 proteins in the individual's spermatozoa. Although this individual exhibited normal semen parameters, his spermatozoa showed impaired penetration ability, deficient hyperactivation, and did not respond to progesterone, in terms of monovalent current potentiation, [Ca2+]i increase, penetration ability enhancement and acrosome reaction inducement, which may explain the individual's idiopathic infertility. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Our novel findings require more cases to support the CATSPER2 CNV identified in this study as a common cause of idiopathic male infertility in patients with normal semen parameters. Therefore, caution must be taken when extrapolating the use of this CNV as a potential biomarker for idiopathic male infertility. WIDER IMPLICATIONS OF THE FINDINGS: The findings from the unique human CATSPER 'knockout' model in this study not only confirm the essential roles of CATSPER in mediating progesterone response and regulating hyperactivation in human spermatozoa but also reveal that disruption of CATSPER current is a significant factor causing idiopathic male infertility. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by National Natural Science Foundation of China (81771644 and 31400996 to T.L.; 31230034 to X.Z.); National Basic Research Program of China (973 Program, 2015CB943003 to X.Z.); National Key Research and Development Program of China (2016YFC1000905 to T.L.); Natural Science Foundation of Jiangxi, China (20121BBG70021 and GJJ12015 to X.Z.; 20161BAB204167 and 20171ACB21006 to T.L.) and the open project of National Population and Family Planning Key Laboratory of Contraceptives and Devices Research (No. 2016KF07 to T.L.). The authors have no conflicts of interest to declare.

A novel cross-species inhibitor to study the function of CatSper Ca2+ channels in sperm.

BACKGROUND AND PURPOSE: Sperm from many species share the sperm-specific Ca2+ channel CatSper that controls the intracellular Ca2+ concentration and, thereby, the swimming behaviour. A growing body of evidence suggests that the mechanisms controlling the activity of CatSper and its role during fertilization differ among species. A lack of suitable pharmacological tools has hampered the elucidation of the function of CatSper. Known inhibitors of CatSper exhibit considerable side effects and also inhibit Slo3, the principal K+ channel of mammalian sperm. The compound RU1968 was reported to suppress Ca2+ signaling in human sperm by an unknown mechanism. Here, we examined the action of RU1968 on CatSper in sperm from humans, mice, and sea urchins. EXPERIMENTAL APPROACH: We resynthesized RU1968 and studied its action on sperm from humans, mice, and the sea urchin Arbacia punctulata by Ca2+ fluorimetry, single-cell Ca2+ imaging, electrophysiology, opto-chemistry, and motility analysis. KEY RESULTS: RU1968 inhibited CatSper in sperm from invertebrates and mammals. The compound lacked toxic side effects in human sperm, did not affect mouse Slo3, and inhibited human Slo3 with about 15-fold lower potency than CatSper. Moreover, in human sperm, RU1968 mimicked CatSper dysfunction and suppressed motility responses evoked by progesterone, an oviductal steroid known to activate CatSper. Finally, RU1968 abolished CatSper-mediated chemotactic navigation in sea urchin sperm. CONCLUSION AND IMPLICATIONS: We propose RU1968 as a novel tool to elucidate the function of CatSper channels in sperm across species.

Exposure to Cadmium Impairs Sperm Functions by Reducing CatSper in Mice.

BACKGROUND: Cadmium (Cd), a common environmental heavy metal and endocrine disruptor, is known to exert toxic effects on the testes. However, the mechanisms accounting for its toxicity in mature spermatozoa remain unclear. METHODS: Adult male C57BL/6 mice were orally administered with CdCl2 for 5 weeks at 3 mg·kg-1·day-1. Additionally, mouse spermatozoa were incubated in vitro with different doses of CdCl2 (0, 10, 50, 250 µM). Several sperm functions including the sperm motility, viability and acrosome reaction (AR) ratio were then examined. Furthermore, the current and expression levels of both the sperm-specific Ca2+ channel (CatSper) and the sperm-specific K+ channel (KSper) were evaluated by patch-clamping and western blotting, respectively. RESULTS: Our data showed that the motility, viability and AR of sperm exposed to cadmium significantly decreased in vivo and in vitro. Interestingly, these changes were correlated with changes in CatSper but not KSper. CONCLUSION: The findings indicate sperm dysfunction during both chronic and acute cadmium exposure as well as a specific role for CatSper in the reproductive toxicity of cadmium.

Bisphenol A Impairs Mature Sperm Functions by a CatSper-Relevant Mechanism.

Bisphenol A (BPA), an endocrine-disrupting chemical, is widely used in the manufacture of daily necessities. Previous studies showed that BPA could impair spermatogenesis. However, its effects on mature spermatozoa are not well known. We aimed to investigate the in vivo and in vitro toxicity of BPA on mature mouse spermatozoa. Different doses of BPA (0, 10, 50, and 250 µg·kg(-1)·d(-1)) were administrated orally to C57BL/6 mice for 8 weeks. Subsequently, the sperm viability, motility, acrosome reaction (AR) ratio together with the expression/current levels of the sperm-specific Ca(2+ )channel (CatSper) and K(+ )channel (KSper) were examined. These parameters were also evaluated after applying BPA directly to normal mouse sperm to appraise the toxicity of BPA to mature sperm in vitro Significant decreases in sperm motility and AR were found in BPA administrated mice, possibly resulting from a BPA caused CatSper down-regulation which was supported by both western blot and patch clamping results. Moreover, direct application of BPA to spermatozoa inhibited CatSper transiently and also caused significant reductions in sperm total motility and AR ratio. In conclusion, both in vivo administration and in vitro application of BPA impair mature sperm functions by a CatSper-relevant mechanism.

SLO3 auxiliary subunit LRRC52 controls gating of sperm KSPER currents and is critical for normal fertility.

Following entry into the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that results in competence to fertilize ova. Associated with capacitation is an increase in membrane conductance to both Ca(2+) and K(+), leading to an elevation in cytosolic Ca(2+) critical for activation of hyperactivated swimming motility. In mice, the Ca(2+) conductance (alkalization-activated Ca(2+)-permeable sperm channel, CATSPER) arises from an ensemble of CATSPER subunits, whereas the K(+) conductance (sperm pH-regulated K(+) current, KSPER) arises from a pore-forming ion channel subunit encoded by the slo3 gene (SLO3) subunit. In the mouse, both CATSPER and KSPER are activated by cytosolic alkalization and a concerted activation of CATSPER and KSPER is likely a common facet of capacitation-associated increases in Ca(2+) and K(+) conductance among various mammalian species. The properties of heterologously expressed mouse SLO3 channels differ from native mouse KSPER current. Recently, a potential KSPER auxiliary subunit, leucine-rich-repeat-containing protein 52 (LRRC52), was identified in mouse sperm and shown to shift gating of SLO3 to be more equivalent to native KSPER. Here, we show that genetic KO of LRRC52 results in mice with severely impaired fertility. Activation of KSPER current in sperm lacking LRRC52 requires more positive voltages and higher pH than for WT KSPER. These results establish a critical role of LRRC52 in KSPER channels and demonstrate that loss of a non-pore-forming auxiliary subunit results in severe fertility impairment. Furthermore, through analysis of several genotypes that influence KSPER current properties we show that in vitro fertilization competence correlates with the net KSPER conductance available for activation under physiological conditions.

Matrine inhibits mouse sperm function by reducing sperm [Ca2+]i and phospho-ERK1/2.

BACKGROUND: Matrine is a bioactive alkaloid that has a variety of pharmacological effects and is widely used in Chinese medicine. However, its effects on male reproduction are not well known. In this study, we aimed to investigate the in vitro toxicity of matrine on mature mouse sperm. METHODS: Mouse cauda epididymal sperm were exposed to matrine (10-200 µM) in vitro. The viability, motility, capacitation, acrosome reaction and fertilization ability of the mouse sperm were examined. Furthermore, the intracellular calcium concentration ([Ca(2+)]i), calcium (Catsper) and potassium (Ksper) currents, and phosphorylation of extracellular signal regulated kinases 1/2 (p-ERK1/2) of the sperm were analyzed. RESULTS: After exposure to 100 µM or more of matrine, mouse cauda epididymal sperm exhibited a significant reduction in total motility, progressive motility, linear velocity and acrosome reaction rate induced by Ca(2+) ionophore A23187. As a result, the fertilization ability of mouse sperm was remarkably decreased by matrine. Our data further demonstrated that matrine significantly reduced sperm [Ca(2+)]i and [Ca(2+)]i-related p-ERK1/2; however, both the CatSper and KSper currents, which are thought to interactively regulate Ca(2+) influx in sperm, were not affected by matrine. CONCLUSION: Our findings indicate that matrine inhibits mouse sperm function by reducing sperm [Ca(2+)]i and suppressing the phosphorylation of ERK1/2.

Emodin inhibits human sperm functions by reducing sperm [Ca(2+)]i and tyrosine phosphorylation.

Emodin, a bioactive anthraquinone widely used in Chinese traditional medicine, disrupts mouse testicular gene expression in vivo. In this study, we investigated the toxicity of emodin to human sperm in vitro. Different doses of emodin (25, 50, 100, 200 and 400µM) were applied to ejaculated human sperm. The results indicated that 100, 200 and 400µM emodin significantly inhibited the total motility, progressive motility and linear velocity of human sperm. In addition, sperm's ability to penetrate viscous medium together with progesterone induced capacitation and acrosome reaction was also adversely affected by emodin. In contrast, emodin did not affect sperm viability. Furthermore, intracellular Ca(2+) concentration ([Ca(2+)]i) and tyrosine phosphorylation, which serve as key regulators of sperm function, were dose-dependently reduced by emodin (50-400µM). These results suggest that emodin inhibits human sperm functions by reducing sperm [Ca(2+)]i and suppressing tyrosine phosphorylation in vitro.

Simultaneous knockout of Slo3 and CatSper1 abolishes all alkalization- and voltage-activated current in mouse spermatozoa.

During passage through the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that renders sperm competent to produce fertilization. Capacitation involves a sequence of changes in biochemical and electrical properties, the onset of a hyperactivated swimming behavior, and development of the ability to undergo successful fusion and penetration with an egg. In mouse sperm, the development of hyperactivated motility is dependent on cytosolic alkalization that then results in an increase in cytosolic Ca(2+). The elevation of Ca(2+) is thought to be primarily driven by the concerted interplay of two alkalization-activated currents, a K(+) current (KSPER) composed of pore-forming subunits encoded by the Kcnu1 gene (also termed Slo3) and a Ca(2+) current arising from a family of CATSPER subunits. After deletion of any of four CATSPER subunit genes (CATSPER1-4), the major remaining current in mouse sperm is alkalization-activated KSPER current. After genetic deletion of the Slo3 gene, KSPER current is abolished, but there remains a small voltage-activated K(+) current hypothesized to reflect monovalent flux through CATSPER. Here, we address two questions. First, does the residual outward K(+) current present in the Slo3 (-/-) sperm arise from CATSPER? Second, can any additional membrane K(+) currents be detected in mouse sperm by patch-clamp methods other than CATSPER and KSPER? Here, using mice bred to lack both SLO3 and CATSPER1 subunits, we show conclusively that the voltage-activated outward current present in Slo3 (-/-) sperm is abolished when CATSPER is also deleted. Any leak currents that may play a role in setting the resting membrane potential in noncapacitated sperm are likely smaller than the pipette leak current and thus cannot be resolved within the limitation of the patch-clamp technique. Together, KSPER and CATSPER appear to be the sole ion channels present in mouse sperm that regulate membrane potential and Ca(2+) influx in response to alkalization.

Sperm-specific ion channels: targets holding the most potential for male contraceptives in development.

There is a global need for an ideal method of male contraception. However, the development of male contraceptives has not been well successful. Research on sperm-specific ion channels, especially the recent advance obtained from electrophysiological studies, has emphasized the conception that those channels are targets with the most potential to develop non-hormonal male contraceptives. While summarizing the general options for male contraception, this review focuses on the properties and functions of sperm ion channels together with the attempts of utilizing these channels to develop male contraceptives. We believe that a deeper insight into the signaling and molecular mechanisms by which ion channels regulate sperm functions will pave the way for developing novel male-based contraceptives.